H-Ras/mitogen-activated protein kinase pathway inhibits integrin-mediated adhesion and induces apoptosis in osteoblasts

Abstract

We have studied the relevance of H-Ras and its downstream effectors to osteoblast functions. 1) Purified human osteoblasts highly expressed integrins β1, α4, α5, α6 and the activation epitope of β1. However, these molecules were markedly down-regulated on osteoblasts transfected with expression vector encoding fully activated H-RasV12, H-RasV12 T35S, activating Raf-1/mitogen-activated protein kinase (MAPK), or an active Raf-1 but not on cells having H-RasV12Y40C, a phosphoinositide 3-kinase (PI3K)-binding mutant. 2) Although osteoblasts spontaneously adhered to fibronectin and laminin in β-dependent manner, the expression of H-RasV12 or H-RasV12T35S, but not H-RasV12Y40C, in osteoblasts reduced their adhesion. 3) Osteoblasts bearing H-RasV12, H-RasV12T35S, or Raf-1 failed to proliferate, whereas those with H-RasV12Y40C proliferated well. (4) The up-regulation of Fas and down-regulation of Bcl-2 were observed in osteoblasts expressing H-RasV12, H-RasV12T35S, or Raf-1. (5) Most of the cells having H-RasV12, H-RasV12T35S, or Raf-1 became annexin-V/high/propidium iodide (PI)high or low and terminal deoxynucleotidyl-transferase-mediated dUTP nick-end labeling (TUNEL)high/PIlow after 24 and 72 h incubation, respectively. Thus, we propose that H-Ras signals followed by Raf-1/MAPK pathway but not PI3K not only reduces β1-mediated adhesion of osteoblasts to matrix proteins but induces apoptosis presumably via the Fas up-regulation and Bcl-2 down-regulation.